Stable SNS-595 Compositions and Methods of Preparation

ABSTRACT

Methods of preparing substantially pure SNS-595 substance are disclosed. Also provided are compositions comprising SNS-595 substance that are substantially pure and essentially free of visible particles.

This application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Application No. 61/240,161, filed Sep. 4, 2009, the entiretyof which is incorporated herein by reference.

1. FIELD

Methods are provided for preparing substantially pure(+)-1,4-dihydro-7-[(3S,4S)-3-methoxy-4-(methylamino)-1-pyrrolidinyl]-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid, together with compositions comprising this substance.

2. BACKGROUND

The compound(+)-1,4-dihydro-7-[(3S,4S)-3-methoxy-4-methylamino-1-pyrrolidinyl]-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid, has the following chemical structure:

This compound is also known as SNS-595 or AG-7352. The United StatesAdopted Names Council (USANC) has assigned the name “vosaroxin” to thiscompound.

SNS-595 is known for its anti-tumor activity (see, Tsuzuki et al., J.Med. Chem., 47:2097-2106, 2004 and Tomita et al., J. Med. Chem.,45:5564-5575, 2002). Treatment of various cancers with SNS-595 has beenproposed in the literature, and SNS-595 has shown preclinical activityagainst various cancer cell lines and xenografts. Various dosingregimens for the use of this compound have been reported. For example,see U.S. Patent Application Pub. Nos. 2005-0203120 A1, 2005-0215583 A1,and 2006-0025437 A1, all of which are incorporated herein by referencein their entireties. SNS-595 is presently being tested in clinicaltrials to assess safety and efficacy in human cancer patients, and hasdemonstrated clinical activity in the treatment of acute myeloidleukemia and ovarian cancer.

SNS-595 can be prepared using techniques known to those of ordinaryskill in the art. See, for example, U.S. Pat. No. 5,817,669, issued Oct.6, 1998; Japanese Patent Application No. Hei 10-173986, published Jun.26, 1998; WO 2007/146335, published Dec. 21, 2007; Tsuzuki et al., J.Med. Chem., 47:2097-2106, 2004; and Tomita et al., J. Med. Chem.,45:5564-5575, 2002, all of which are incorporated herein by reference intheir entireties.

International Patent Application No. WO 2007/146335 describes anexemplary process for the synthesis of SNS-595. As shown in Scheme 1,this synthesis proceeds via Intermediate 1, which is reacted withCompound 2 in the presence of a base, such as triethylamine orN,N-diisopropylethylamine to form Compound 3. The ester group ofCompound 3 is subsequently hydrolyzed to afford SNS-595.

The route described in Scheme 1, however, typically yields undesirableamounts of impurities, i.e., by-products of the reactions, which aredifficult to deplete or remove from the SNS-595 drug substance andSNS-595 drug products.

Although certain by-products can exist in SNS-595 preparations, reducingthe amount of these impurities in the drug substance and the final drugproduct is important. Since cancer patients undergo significantchemotherapy and radiation therapy and can, therefore, have compromisedimmune systems, it is beneficial to deliver to these patients drugproduct that is characterized by high purity. Further, for intravenousor intraarterial administration of a drug product, the purity andphysical characteristics of the drug product are important because thedrug product enters directly into the bloodstream.

Thus, there remains a need for improved methods for preparing SNS-595substantially free of contaminants, thereby providing a drug substancein a substantially pure form that is well suited for formulation intopharmaceutical products for the treatment of cancer without the need forlaborious purification steps.

3. SUMMARY

Processes are provided that can yield a substantially pure SNS-595substance. In addition, the processes can be scaled up to commercialmanufacturing of substantially pure SNS-595 substance.

In some embodiments, processes are provided for preparing asubstantially pure SNS-595 substance, comprising:

-   -   (a) reacting Compound 3

-   -   -   with a first aqueous base followed by neutralizing to obtain            a primary SNS-595 hydrate;

    -   (b) dehydrating the primary SNS-595 hydrate from step (a) and        reacting the dehydrated product with a second aqueous base        followed by neutralizing to obtain a secondary SNS-595 hydrate;        and

    -   (c) dehydrating the secondary SNS-595 hydrate obtained in        step (c) to obtain the substantially pure SNS-595 substance.

In some cases, it may be desirable to react the substantially pureSNS-595 substance obtained in step (c) with a further aqueous base,neutralize, and then dehydrate to further improve the purity of thesubstantially pure SNS-595 product. The first aqueous base, the secondaqueous base, and the further aqueous base(s) can be the same ordifferent. Likewise, the acids employed during the neutralization stepsmay be the same or different. Recycling of the substantially pureSNS-595 substance through the described steps of treatment with aqueousbase, neutralization, and dehydration may be performed a plurality oftimes to sequentially further purify the SNS-595 substance until asubstantially pure SNS-595 substance of a desired level of purity isobtained.

In some embodiments, processes are provided for preparing asubstantially pure SNS-595 substance, comprising:

-   -   (a) reacting Compound 1

-   -   with Compound 2

-   -   in the presence of N,N-diisopropylethylamine and water to obtain        substantially pure Compound 3

and

-   -   (b) reacting Compound 3 with an aqueous base to form the        substantially pure SNS-595 substance.

In some embodiments, provided herein are compositions comprisingsubstantially pure SNS-595 substance, wherein the substantially pureSNS-595 substance comprises about 0 to 0.01% Compound 4

at the time of production of the compositions, based on total weight ofthe substantially pure SNS-595 substance.

In some embodiments, provided herein are compositions comprisingsubstantially pure SNS-595 substance, wherein the substantially pureSNS-595 substance comprises about 0 to 0.02% Compound 5

at the time of production of the compositions based on total weight ofthe substantially pure SNS-595 substance.

In some embodiments, compositions are provided that comprise SNS-595 andwater, wherein about 100 mg of SNS-595 is present for every 10 mL of thecompositions, wherein the compositions are essentially free of visibleparticles, and wherein the compositions are stable at, for example, 3,6, 9, 12, or 24 months after production.

In some embodiments, compositions are provided that comprise SNS-595 andwater, wherein about 100 mg of SNS-595 is present for every 10 mL of thecompositions, wherein the compositions are essentially free ofsub-visible particles, and wherein the compositions are stable at, forexample, 3, 6, 9, 12, or 24 months after production.

In some embodiments, the processes described herein are performed on akilogram scale.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the observed relationship between amounts of Compound6 in SNS-595 bulk drug product solutions formulated from drug substanceshaving different amounts of Compound 4.

FIG. 2 illustrates the observed relationship between the amount ofCompound 4 in SNS-595 drug substance and the amount of residual Compound1 in Compound 3 used to prepare the drug substance.

FIG. 3 illustrates the observed relationship between the amount ofCompound 5 in SNS-595 drug substance and the amount of residual Compound1 in Compound 3 used to prepare the drug substance.

5. DETAILED DESCRIPTION 4.1 Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications mentioned herein are incorporated by reference in theirentirety.

As used herein, “SNS-595” refers to(+)-1,4-dihydro-7-[(3S,4S)-3-methoxy-4-methylamino-1-pyrrolidinyl]-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid, as well as any ionic form, salts, solvates, e.g., hydrates, orother forms of that compound, including mixtures thereof. Thus,compositions comprising SNS-595 include(+)-1,4-dihydro-7-[(3S,4S)-3-methoxy-4-methylamino-1-pyrrolidinyl]-4-oxo-1-(2-thiazolyl)-1,8-naphthyridine-3-carboxylicacid or, in some embodiments, an ionic form thereof, salt, solvate,e.g., hydrate, polymorph, pseudomorph, or other form of the compound. Insome embodiments, SNS-595 is provided as a pharmaceutically acceptablesalt. SNS-595 is also referred to as AG-7352, voreloxin, and vosaroxin.

As used herein, “substantially pure SNS-595 substance” refers to acomposition consisting essentially of SNS-595, i.e., comprising lessthan about 1.0% of any other individual compound or impurity based ontotal weight of the composition (wt %). For example, in someembodiments, such compositions comprise about 0 to 0.5%, about 0 to0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to 0.02%, or about 0to 0.01% Compound 4 based on total weight of the composition.

In some embodiments, such compositions consist essentially of 0 to 0.5%,0 to 0.1%, 0 to 0.05%, 0 to 0.03%, 0 to 0.02%, or 0 to 0.01% Compound 4based on total weight of the composition. In some embodiments, suchcompositions have 0 to 0.5%, 0 to 0.1%, 0 to 0.05%, 0 to 0.03%, 0 to0.02%, or 0 to 0.01% Compound 4 based on total weight of thecomposition. In some embodiments, the compositions have ≦0.02% ofCompound 4 based on total weight of the composition.In some embodiments, such compositions comprise about 0 to 0.04%, about0 to 0.03%, or about 0 to 0.02% Compound 5 based on total weight of thecomposition.

In some embodiments, such compositions consist essentially of 0 to0.04%, 0 to 0.03%, or 0 to 0.02% Compound 5 based on total weight of thecomposition. In some embodiments, such compositions have of 0 to 0.04%,0 to 0.03%, or 0 to 0.02% Compound 5 based on total weight of thecomposition. In some embodiments, the compositions have ≦0.15% ofCompound 5 based on total weight of the composition.Other compositions that are substantially pure SNS-595 substances aredescribed herein. In some embodiments, such compositions comprise about0 to 0.5%, about 0 to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0to 0.02%, or about 0 to 0.01% Compound 6 based on total weight of thecomposition.

In some embodiments, such compositions consist essentially of about 0 to0.5%, about 0 to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to0.02%, or about 0 to 0.01% Compound 6 based on total weight of thecomposition. In some embodiments, such compositions have about 0 to0.5%, about 0 to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to0.02%, or about 0 to 0.01% Compound 6 based on total weight of thecomposition.In some embodiments, such compositions comprise about 0 to 0.5%, about 0to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to 0.02%, or about0 to 0.01% Compound 7 based on total weight of the composition.

In some embodiments, such compositions consist essentially of about 0 to0.5%, about 0 to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to0.02%, or about 0 to 0.01% Compound 7 based on total weight of thecomposition. In some embodiments, such compositions have about 0 to0.5%, about 0 to 0.1%, about 0 to 0.05%, about 0 to 0.03%, about 0 to0.02%, or about 0 to 0.01% Compound 7 based on total weight of thecomposition. In some embodiments, the compositions have ≦0.15% ofCompound 7 based on total weight of the composition.

As used herein, “SNS-595 substance” refers to a composition comprisingSNS-595 and one or more other compounds. In some embodiments, theSNS-595 substance comprises SNS-595 and Compound 4 and/or Compound 5.

As used herein and unless otherwise indicated, “about” refers to up toplus or minus 10% of the indicated value. For example, “about 0.01%”refers to 0.009% to 0.011%, “about 25° C.” refers to 22.5° C. to 27.5°C., and “about 0.6 M” refers to 0.54 M to 0.66 M. In some embodiments,about refers to up to plus or minus 9, 8, 7, 6, 5, 4, 3, 2, or 1% of theindicated value. Similarly, for a range of values, use of “about” refersto both the upper limit and the lower limit of the stated range.

The term “about” with respect to a pH value is intended to mean that theacceptable error for that pH value is no greater than 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pH unit. In certainembodiments, the error for a pH value is no greater than 0.02 pH unit(see, Method 791 in USP XXVI (2003), incorporated herein by reference inits entirety).

As used herein, “aqueous base” refers to any aqueous solution of one ormore bases, which, in some embodiments, are one or more strong bases(pKa>13). Examples of strong bases include, without limitation,hydroxides of alkali and alkaline earth metals or ammonium hydroxide.Aqueous bases may be aqueous solutions of organic or inorganic bases. Insome embodiments, the base is provided as an aqueous solution ofpotassium hydroxide, lithium hydroxide, sodium hydroxide, or ammoniumhydroxide. In some embodiments, the aqueous base has a molarconcentration of about 0.6-1.1 M. In some embodiments, the aqueous baseis an aqueous solution of a hydroxide having a molar concentration ofabout 0.6, 0.7, 0.8, 0.9, 1.0, or about 1.1 M. In some embodiments, thebase may be provided in solid form. In some embodiments, the solid formis a pellet or a powder.

As used herein and unless otherwise indicated, the term “hydrate” meansa compound or salt thereof, further including a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces. As used herein and unless otherwise indicated, the term“solvate” means a solvate formed from the association of one or moresolvent molecules to a compound provided herein. The term “solvate”includes hydrates (e.g., monohydrate, dihydrate, trihydrate,tetrahydrate, and the like). The solvates of SNS-595 can be crystallineor non-crystalline.

As used herein, “SNS-595 hydrate” refers to SNS-595 having astoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces. In some embodiments, the SNS-595hydrate is crystalline or non-crystalline. In some embodiments, theSNS-595 hydrate comprises about 0.8 to 1.2 molar equivalents of waterper mole of SNS-595. In some embodiments, the SNS-595 hydrate comprisesabout 1 molar equivalent of water per mole of SNS-595.

As used herein, “dehydrating” refers to removing water bound to theSNS-595 of a SNS-595 hydrate. Dehydrating methods are known to those ofordinary skill in the art. In some embodiments, dehydrating isaccomplished by contacting the SNS-595 hydrate with a compound capableof removing water bound to the SNS-595 of the SNS-595 hydrate. Suchcompounds include dehydration solvents. In some embodiments, the solventis hygroscopic and/or protic. Exemplary solvents include, withoutlimitation, methanol, ethanol, isopropanol, acetone, or others apparentto those of ordinary skill in the art. In some embodiments, the solventis anhydrous. In some embodiments, the solvent is anhydrous ethanol. Insome embodiments, the anhydrous ethanol comprises less than 0.5% water.In a particular embodiment, dehydrating is accomplished by contactingSNS-595 hydrate with anhydrous ethanol at about 25-80° C., about 40-80°C., about 60-80° C., or about 80° C. In some cases, dehydration may beaccomplished by thermal means in the absence of a solvent.

The amount of water in a hydrate may be analyzed using a number oftechniques as understood in the art. For example, the amount of watermay be determined based on the observed weight loss in athermogravimetric analysis (TGA) thermogram. In addition, the exhaustfrom a TGA furnace may be coupled to an instrument of chemical analysis,such as a mass spectrometry instrument or an infrared spectroscopyinstrument, to confirm the chemical purity of the water vapor emittedupon heating. Water loss may also be quantified by direct gravimetricmeans such as “Loss on Dying <731>” as described in the U.S.Pharmacopoeia, the entirety of which is incorporated herein byreference. Karl Fischer (KF) analysis may be used to analyze the watercontent of a hydrate sample. Coulometric KF analysis for waterdetermination may be performed using a Mettler Toledo DL39 Karl Fischertitrator or other equipment. In one method, approximately 14-32 mg of asample is placed in a KF titration vessel containing HYDRANAL®-CoulomatAD reagent for coulometric KF titration and mixed for 60 seconds toensure dissolution. The sample is then titrated by means of a generatorelectrode, which produces iodine by electrochemical oxidation. Theanalysis is repeated to ensure reproducibility of the measurements.

As used herein, “neutralizing” or “neutralization” refers to the processof adjusting the pH of a solution to neutrality or approximateneutrality, e.g., pH of from 6.0 to 8.0, 7.0 to 8.0, or 7.3 to 7.7.

As used herein, “particulate matter” refers to any matter formed as aresult of by-products of a synthetic preparation of SNS-595 that isinsoluble or partially soluble in water or an aqueous mixture. In someembodiments, the particulate matter comprises visible particles. Otherimpurities including particulates such as lint, glass, metal, and thelike may be present in the SNS-595 compositions at or below levelspermitted for administration to human subjects in the treatment ofdisease.

As used herein, “visible particles” refers to insoluble or partiallysoluble solids in a liquid solution, e.g., an aqueous solution, that arevisible to a human eye. In some embodiments, the visible particles arevisible under natural sunlight, white-light, fluorescent lighting, orincandescent lighting. In further embodiments, the visible particles arevisible when observed under lighting having an intensity of 600-7000lux, 900-4000 lux, 850-4650 lux, or 2000-3750 lux. In some embodiments,the particles are visible when inspected for about 1-60, 1-30, 1-15,1-10, 1-5, or 5 seconds.

In a particular embodiment, the visible particles are visible whentested according to the method described by European Pharmacopeia 5.0,Section 2.9.20, the entirety of which is incorporated herein byreference. In this method, an apparatus having a viewing stationcomprising (1) a matt black panel of appropriate size held in a verticalposition; (2) a non-glare white panel of appropriate size held in avertical position next to the black panel; and (3) an adjustablelampholder fitted with a suitable, shaded, white-light source and with asuitable light diffuser (e.g., a viewing illuminator containing two 13 Wfluorescent tubes, each 525 mm in length) is used. The intensity ofillumination at the viewing point is maintained between 2000 lux and3750 lux. Higher values may be used for glass and plastic containers.Adherent labels are removed from the container containing the sample tobe tested. The outside of the container is washed and dried. Thecontainer is gently swirled or inverted while ensuring that air bubblesare not introduced, and the container is observed for about 5 seconds infront of the white panel to determine whether visible particles arepresent. The container is then observed for about 5 seconds in front ofthe black panel to determine whether visible particles are present. Insome embodiments, the visible particles are visible against a whitepanel. In some embodiments, the visible particles are visible against ablack panel.

In some embodiments the visible particles have an average diameter of atleast 50 μm, at least 75 μm, at least 100 μm, at least 150 μm, or atleast 200 μm. In some embodiments the visible particles have an averagediameter of about 50-500 μm, about 50-300 μm, about 100-500 μm, or about100-300 μm.

As used herein, “sub-visible particles” refers to particulate matterdetectable by the Light Obscuration Particle Count Test or MicroscopicParticle Count Test described in the U.S. Pharmacopoeia, <788>Particulate Matter in Injections, which is incorporated herein byreference in its entirety.

In some embodiments, the visible and/or sub-visible particles comprisematter from the production process of a drug substance composition,including, without limitation, the synthesis or formulation of the drugsubstance, or the packaging of the composition. In some embodiments, thevisible particles comprise metal, glass, lint, or the like.

In certain embodiments, the visible and/or sub-visible particlescomprise one or more of Compound 5

and/or Compound 6

and/or Compound 7

As used herein, “essentially free of visible particles” refers to aliquid, e.g., aqueous, composition comprising SNS-595 that does notcontain visible particles. In some embodiments, the composition does notcontain visible particles when adjudged by the methods described byEuropean Pharmacopeia 5.0, Section 2.9.20 discussed above.

As used herein, “essentially free of sub-visible particles” refers to aliquid, e.g., aqueous, composition comprising SNS-595 that does notcontain sub-visible particles.

As used herein, “stable” refers to a composition comprising SNS-595that, when stored in a container or vial, can remain essentially free ofvisible or sub-visible particles, or can maintain a specified amount ofvisible and/or sub-visible particles, for a specified period of time,e.g., 1, 3, 6 or 9 months. For example, an aqueous composition that isessentially free of visible particles and stable for 6 months withrespect to visible particles refers to an aqueous composition that isjudged to be essentially free of visible particles at any point in thetime during the period starting from a referenced starting point, e.g.,the time when the composition was produced and/or added to a container)to 6 months after the composition was prepared. In addition, if thestable composition is a bulk composition, then that composition iscapable of being distributed into a lot of containers, e.g., vials,wherein any container containing the distributed composition that isessentially free of visible particles at the time of production of thelot is capable of remaining essentially free of visible particles forthe specified period of time, e.g., 1, 3, 6 or 9 months. In someembodiments, the composition is stable for at least 1, 2, 4, 6, 8, 10,12, 15, 20, or 25 days. In some embodiments, the composition is stablefor at least 1, 3, 6, 9, 12, 18, 24, 36, 42, or 48 months. In someembodiments, the composition is stable starting from the time ofproduction.

As used herein, “time of production” refers to the point in time whenthe SNS-595 composition or container comprising SNS-595 is produced. Insome embodiments, “time of production” is the time when the desiredamounts of substantially pure SNS-595 substance, sorbitol, water, andmethanesulfonic acid are mixed. In other embodiments, the “time ofproduction” is the time when the desired amounts of substantially pureSNS-595 substance, sorbitol, water, and methanesulfonic acid are mixedand added to a container, e.g., vial. In some embodiments, the time isless than 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day afterthe composition or container comprising SNS-595 is produced. In someembodiments, the time is less than 20, 16, 12, 8, 4, or 2 hours afterthe composition or container comprising SNS-595 is produced.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” includes, but is not limited to, asalt of an acidic or basic group that can be present in the compoundsprovided herein. Under certain acidic conditions, the compound can forma wide variety of salts with various inorganic and organic acids. Theacids that can be used to prepare pharmaceutically acceptable salts ofsuch basic compounds are those that form salts comprisingpharmacologically acceptable anions including, but not limited to,acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate, carbonate, chloride, bromide, iodide,citrate, dihydrochloride, edetate, edisylate, estolate, esylate,fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,hexylresorcinate, hydrabamine, hydroxynaphthoate, isethionate, lactate,lactobionate, malate, maleate, mandelate, methanesulfonate (mesylate),methylsulfate, muscate, napsylate, nitrate, pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,succinate, sulfate, tannate, tartrate, teoclate, triethiodide, andpamoate. Under certain basic conditions, the compound can form basesalts with various pharmacologically acceptable cations. Non-limitingexamples of such salts include alkali metal or alkaline earth metalsalts and, particularly, calcium, magnesium, sodium, lithium, zinc,potassium, and iron salts.

As used herein, “reprocessing” refers to subjecting the SNS-595(typically obtained from the saponification of Compound 3) tosaponification conditions a second time or more. In some embodiments,the SNS-595 is reprocessed more than one time. In some embodiments, thesaponification conditions utilize aqueous sodium hydroxide, ethanol, andacetic acid. In some embodiments, the saponification conditions comprisereacting Compound 3 with an aqueous base followed by neutralizing toform SNS-595 hydrate and dehydrating the SNS-595 hydrate.

As used herein, “substantially pure Compound 3” refers a compositionconsisting essentially of Compound 3

In some embodiments, substantially pure Compound 3 compositions maycomprise 0 to 0.3%, 0 to 0.25%, 0 to 0.2%, 0 to 0.1%, 0 to 0.05%, or 0to 0.01% Compound 1, based on total weight of the composition.

In some embodiments, substantially pure Compound 3 compositions mayconsist essentially of 0 to 0.3%, 0 to 0.25%, 0 to 0.2%, 0 to 0.1%, 0 to0.05%, or 0 to 0.01% Compound 1, based on total weight of thecomposition. In some embodiments, substantially pure Compound 3compositions may have 0 to 0.3%, 0 to 0.25%, 0 to 0.2%, 0 to 0.1%, 0 to0.05%, or 0 to 0.01% Compound 1, based on total weight of thecomposition. In some embodiments, such compositions comprise 0 to 0.1%,or 0 to 0.05%, Compound 1 based on total weight of the composition.

The purity of substantially pure SNS-595 substance or substantially pureCompound 3 provided herein, as well as the amounts of other compoundsmentioned herein, can be determined by standard methods of analysis usedby those of ordinary skill in the art, such as high performance liquidchromatography (HPLC). In this application, reference to a compositionhaving “0” of any component means that at least the measured amount ofthat component is lower than the limit of detection using suchanalytical methods or is 0.001% w/w or less.

4.2 Methods of Preparation

International Patent Application No. WO 2007/146335 and U.S. ProvisionalApplication No. 61/141,856 describe exemplary processes known in the artfor the synthesis of the SNS-595 compound. As shown in Scheme 2, thissynthesis proceeds via Intermediate 1, which is reacted with Compound 2in the presence of triethylamine or N,N-diisopropylethylamine to formCompound 3. The ester group of Compound 3 is subsequently hydrolyzed toafford SNS-595.

However, when this process is followed, several impurities can beformed. Specifically, as shown in Scheme 3, following saponification ofCompound 3 in basic conditions, approximately 0.04% of Compound 4 and<0.05% of Compound 5 can be observed in the reaction product, inaddition to SNS-595.

Without being limited to any theory, Compound 5 may be caused by thepresence of Compound 1 during the saponification step. Indeed, as shownin Scheme 4, the product isolated from the reaction of Compounds 1 and 2in the presence of triethylamine or N,N-diisopropylethylamine cancontain approximately 0.3-0.6% by weight residual Compound 1.

Without being bound to any theory, Compound 4 may be the result of areaction between Compound 1 and hydroxide, possibly through a1,4-addition of water. Indeed, when Compound 1 is treated with aqueoussodium hydroxide, Compound 4 can be obtained, in addition to Compound 5,illustrated in Scheme 5.

4.2(a) Reprocessing Method

In certain embodiments, processes are provided for preparingsubstantially pure SNS-595 substance by reprocessing a SNS-595substance.

As noted, the reaction of Compound 1 with Compound 2 can result in theformation of a mixture of Compound 3 and residual Compound 1. Subsequenttreatment of this mixture with aqueous base, i.e., saponificationconditions, can result in the formation of a mixture of SNS-595,Compound 4, and Compound 5. However, when this mixture is againsubjected to the saponification conditions, i.e., reprocessed, theresulting product can contain SNS-595 with lesser amounts of Compound 4,as shown in Scheme 6.

Additional experiments show that several cycles of such reprocessing maybe used to sequentially further reduce the residual amount of Compound 4in the composition. In some embodiments, the reprocessing is performed anumber of times sufficient to reduce the level of Compound 4 to belowthe limit of detection by conventional methods, such as those disclosedherein or in the art.

In certain embodiments, reprocessing is accomplished by solubilizingSNS-595 hydrate with an aqueous base followed by neutralizing with acid,followed by dehydration.

In certain embodiments, provided herein are processes for preparingsubstantially pure SNS-595 substance comprising:

-   -   (a) reacting Compound 3

-   -   -   with a first aqueous base followed by neutralizing to obtain            a primary SNS-595 hydrate;

    -   (b) dehydrating the primary SNS-595 hydrate from step (a) and        reacting the dehydrated product with a second aqueous base        followed by neutralizing to obtain a secondary SNS-595 hydrate;        and

    -   (c) dehydrating the secondary SNS-595 hydrate obtained in        step (b) to obtain substantially pure SNS-595 substance.

The first and second aqueous bases may be the same or different. In someembodiments, the first and second aqueous bases of steps (a) and (b) areeach, independently, potassium hydroxide, sodium hydroxide, or lithiumhydroxide. In some embodiments, the first and second aqueous bases ofsteps (a) and (b) are each, independently, sodium hydroxide or lithiumhydroxide. In some embodiments, the first and second aqueous bases areeach sodium hydroxide. In some embodiments, the first or second aqueousbase, each independently, has a molar concentration of about 0.6-1.1 M.In some embodiments, the first or second aqueous base is an aqueoussolution of a hydroxide, wherein the first or second aqueous base eachindependently has a molar concentration of about 0.6, 0.7, 0.8, 0.9,1.0, or about 1.1 M.

The dehydration steps, independently, may be performed by treatment withdehydration solvents known to those of ordinary skill in the art. Forexample, dehydration may be accomplished by treating the material to bedehydrated with a hygroscopic and/or protic solvent. Exemplary solventsinclude, without limitation, ethanol, methanol, isopropanol, andacetone. In some embodiments, the dehydration solvent is anhydrous.

In some embodiments, the SNS-595 hydrate of step (b) or (c) isdehydrated with ethanol. In some embodiments, the ethanol is anhydrous.In particular embodiments, the anhydrous ethanol comprises less than0.5% water. In some embodiments, the secondary SNS-595 hydrate of step(c) is dehydrated with anhydrous ethanol at a temperature of about25-80° C., about 40-80° C., about 60-80° C., or about 80° C.

Neutralization steps, independently, may be accomplished with any acidknown to those of ordinary skill in the art. Inorganic acids, organicacids, or combinations thereof may be utilized. Acids may also beaqueous. Exemplary acids include, without limitation, acetic acid,hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid,carbonic acid, phosphoric acid, oxalic acid, or nitric acid. In someembodiments, neutralization is accomplished with acetic acid. In someembodiments, the neutralization step adjusts the pH to 6.0 to 8.0. Incertain embodiments, pH is adjusted to 7.0 to 8.0. In certainembodiments, pH is adjusted to about 7.3 to 7.7.

In certain embodiments, the first and second aqueous bases of steps (a)and (b) are each sodium hydroxide; pH is adjusted to about 7.3 to 7.7with acetic acid during neutralization; steps (a) and (b) are performedin the presence of ethanol; and dehydration steps are accomplished withanhydrous ethanol at a temperature between about 25-80° C.

The aqueous base reaction and neutralization may be performed in thepresence of organic solvents known to those of ordinary skill in theart. In certain embodiments, the solvent is capable of dissolvingSNS-595-hydrate when treated with aqueous base and, at the same time,capable of precipitating SNS-595 hydrate after subsequentneutralization. In some embodiments, step (a) or step (b) is performedin the presence of ethanol or methanol. In some embodiments, step (a) orstep (b) is performed in the presence of ethanol. In some embodiments,step (a) and step (b) are each performed in the presence of ethanol. Thevolume of ethanol or methanol suitable to carry out steps (a) or (b) maybe readily determined by those of ordinary skill in the art. In certainembodiments, step (a) or step (b) is performed in the presence ofethanol or methanol, wherein the aqueous base is about 1 to about 20%ethanol or methanol by volume. In particular embodiments, the aqueousbase is about 3, 5, 10, or 15% ethanol by volume.

In some embodiments, the substantially pure SNS-595 substance obtainedfrom step (c) comprises about 0 to 0.03% Compound 4 based on totalweight of substantially pure SNS-595 substance. In some embodiments, thesubstantially pure SNS-595 substance obtained from step (c) comprisesabout 0 to 0.02% Compound 4 based on total weight of substantially pureSNS-595 substance. In some embodiments, the substantially pure SNS-595substance obtained from step (c) comprises about 0 to 0.01% Compound 4based on total weight of substantially pure SNS-595 substance. In someembodiments, the substantially pure SNS-595 substance obtained from step(c) comprises about 0 to 0.03%,0 to 0.02%, or 0 to 0.01% Compound 4based on total weight of substantially pure SNS-595 substance.

Compound 3 can be obtained by the reaction of Compound 1

with Compound 2

Compound 1 can be prepared or obtained by any source or method deemedsuitable by those of ordinary skill in the art. Exemplary methods aredescribed in WO 2007/146335, which is incorporated herein by referencein its entirety.

In some embodiments, Compound 3 is present as a mixture that comprisesabout 0 to 0.7% of Compound 1, based on total weight of the mixture. Insome embodiments. Compound 3 is present as a mixture that comprisesabout 0 to 0.6% of Compound 1, based on total weight of the mixture. Insome embodiments, Compound 3 is present as a mixture that comprisesabout 0 to 0.3% of Compound 1, based on total weight of the mixture. Insome embodiments, Compound 3 is present as a mixture that has 0 to 0.7%,0 to 0.6%, or 0 to 0.3% Compound 1 based on total weight of the mixture.In some embodiments, Compound 3 is present as a mixture that comprises 0to 0.7%, 0 to 0.6%, or 0 to 0.3% Compound 1 based on total weight of themixture. In some embodiments, Compound 3 is present as a mixture thatconsists essentially of 0 to 0.7%, 0 to 0.6%, or 0 to 0.3% Compound 1based on total weight of the mixture.

4.2(b) Wet N,N-Diisopropylethylamine Method

In certain embodiments, Compound 1 can be reacted with Compound 2 in thepresence of N,N-diisopropylethylamine (DIPEA) and water in acetonitrileto obtain substantially pure Compound 3, which can be subsequentlyreacted with an aqueous base to obtain substantially pure SNS-595substance.

When Compound 1 is reacted with Compound 2 in the presence of DIPEA andwater in acetonitrile, it can be observed that Compound 3 may beobtained with lower levels of Compound 1 impurity compared to when thereaction is done in the absence of water. As shown in Scheme 7,subsequent saponification with aqueous base can provide SNS-595 havinglower levels of both Compound 4 and Compound 5 compared to reaction ofCompound 1 with Compound 2 in DIPEA in the absence of water.

In some embodiments, Compound 3

is prepared by reacting Compound 1

with Compound 2

in DIPEA and water to obtain Compound 3.

In some embodiments, 0.5% to 10% water in acetonitrile is used. In someembodiments, 2% to 8% water in acetonitrile is used. In someembodiments, 4% to 6% water in acetonitrile is used.

In some embodiments, water can be added at the beginning, during, or atthe end of the reaction.

In some embodiments, the process is performed at about 25° C. In someembodiments, the reaction mixture may be heated to effect consumption ofCompound 1. In some embodiments, the process is performed initially at atemperature of about 25° C. and subsequently at a higher temperature. Insome embodiments, the higher temperature is below reflux temperature. Ina particular embodiment, the temperature at the start of the reaction is25° C.; such temperature is maintained for a desired time, after whichthe temperature is raised to about 40-50° C. and maintained at about40-50° C. for a second desired period. In further embodiments, theprocess is performed initially at temperature of about 25° C. for about12 hours (hr) and subsequently at a temperature of about 40-45° C. forabout 3-5 hr.

In some embodiments, the substantially pure Compound 3 obtainedcomprises about 0 to 0.1%, about 0 to 0.05%, or about 0 to 0.03%Compound 1

based on total weight of substantially pure Compound 3. In someembodiments, the substantially pure Compound 3 obtained comprises 0 to0.1%, 0 to 0.05%, or 0 to 0.03% Compound 1.

In some embodiments, provided herein are processes for preparingsubstantially pure SNS-595 substance comprising:

-   -   (a) reacting Compound 1

with Compound 2

in DIPEA and water to obtain substantially pure Compound 3

and

-   -   (b) reacting the substantially pure Compound 3 with an aqueous        base followed by neutralizing to obtain a primary SNS-595        hydrate; and    -   (c) dehydrating the primary SNS-595 hydrate obtained in step (a)        to obtain the substantially pure SNS-595 substance.

In some embodiments, step (a) is performed in the presence ofacetonitrile.

In some embodiments, 0.5% to 10% water in acetonitrile is used. In someembodiments, 2% to 8% water in acetonitrile is used. In someembodiments, 4% to 6% water in acetonitrile is used.

In some embodiments, water can be added at the beginning, during, or atthe end of the reaction.

In some embodiments, the process is performed at about 25° C. In someembodiments, the reaction mixture may be heated to effect consumption ofCompound 1. In some embodiments, the process is performed initially at atemperature of about 25° C. and subsequently at a higher temperature. Insome embodiments, the higher temperature is below reflux temperature. Ina particular embodiment, the temperature at the start of the reaction is25° C.; such temperature is maintained for a desired time, after whichthe temperature is raised to about 40-50° C. and maintained at about40-50° C. for a second desired period. In some embodiments, step (a) isperformed initially at about 25° C. and subsequently at about 40-45° C.In a particular embodiment, the step (a) is performed initially at about25° C. for about 12 hr and subsequently at about 40-45° C. for about 3-5hr. In some embodiments, the step (a) is performed at about 25° C.

In some embodiments, the substantially pure Compound 3 obtainedcomprises about 0 to 0.1%, about 0 to 0.05%, or about 0 to 0.03%Compound 1

based on total weight of the substantially pure Compound 3. In someembodiments, the substantially pure Compound 3 obtained comprises 0 to0.1%, 0 to 0.05%, or 0 to 0.03% Compound 1.

In some embodiments, the aqueous base is potassium hydroxide, sodiumhydroxide, or lithium hydroxide. In some embodiments, the aqueous baseis sodium hydroxide or lithium hydroxide. In some embodiments, theaqueous base is sodium hydroxide. In some embodiments, the aqueous basehas a molar concentration of about 0.6-1.1 M. In some embodiments, theaqueous base is an aqueous solution of a hydroxide having a molarconcentration of about 0.6, 0.7, 0.8, 0.9, 1.0, or about 1.1 M.

In some embodiments, step (b) is performed in the presence of ethanol ormethanol. In some embodiments, step (b) is performed in the presence ofethanol. The volume of ethanol or methanol suitable to carry out step(b) may be readily determined by those of ordinary skill in the art. Incertain embodiments, step (b) is performed in the presence of ethanol ormethanol, wherein the aqueous base is about 1 to about 20% ethanol ormethanol by volume. In particular embodiments, the aqueous base is about3, 5, 10, or about 15% ethanol by volume.

In some embodiments, an acid is added in step (b) after reactingCompound 3 with the aqueous base to adjust the pH to 6.0 to 8.0.Suitable acids include, but are not limited to acetic acid, hydrochloricacid, sulfuric acid, and the like. In certain embodiments, the acid isacetic acid. In certain embodiments, pH is adjusted to about 7.3 to 7.7.

In certain embodiments, the aqueous base of step (b) is sodiumhydroxide; pH is adjusted to about 7.3 to 7.7 with acetic acid afterCompound 3 is reacted with the sodium hydroxide in step (b); and step(b) is performed in the presence of ethanol.

In some embodiments, the primary SNS-595 hydrate of step (c) isdehydrated with ethanol. In some embodiments, the ethanol is anhydrous.In particular embodiments, the anhydrous ethanol comprises less than0.5% water. In some embodiments, the primary SNS-595 hydrate of step (c)is dehydrated with anhydrous ethanol at a temperature of about 25-80°C., about 40-80° C., about 60-80° C., or about 80° C.

In certain embodiments, the substantially pure SNS-595 substanceobtained from step (b) comprises about 0 to 0.02% or about 0 to 0.01%Compound 4

based on total weight of substantially pure SNS-595 substance. In someembodiments, the substantially pure SNS-595 substance obtained from step(b) has 0 to 0.02%, or 0 to 0.01% Compound 4. In some embodiments, thesubstantially pure SNS-595 substance obtained from step (b) consistsessentially of 0 to 0.02%, or 0 to 0.01% Compound 4. In someembodiments, the substantially pure SNS-595 substance obtained from step(b) comprises 0 to 0.02%, or 0 to 0.01% Compound 4.

In some embodiments, the substantially pure SNS-595 substance obtainedfrom step (b) comprises about 0 to 0.02% Compound 5

based on total weight of substantially pure SNS-595 substance. In someembodiments, the substantially pure SNS-595 substance obtained from step(b) has 0 to 0.02% Compound 5. In some embodiments, the substantiallypure SNS-595 substance obtained from step (b) consists essentially of 0to 0.02% Compound 5. In some embodiments, the substantially pure SNS-595substance obtained from step (b) comprises 0 to 0.02% Compound 5.

In some embodiments, the substantially pure SNS-595 substance obtainedin step (b) is reprocessed. In some embodiments, provided herein areprocesses for preparing substantially pure SNS-595 substance comprising:

-   -   (a) reacting Compound 1

with Compound 2

in the presence of DIPEA and water to obtain substantially pure Compound3

-   -   (b) reacting substantially pure Compound 3 with a first aqueous        base followed by neutralizing to obtain a primary SNS-595        hydrate;    -   (c) dehydrating the primary SNS-595 hydrate obtained in step (b)        to form a SNS-595 substance;    -   (d) reacting the SNS-595 substance from step (c) with a second        aqueous base followed by neutralizing to obtain a secondary        SNS-595 hydrate; and    -   (e) dehydrating the secondary SNS-595 hydrate obtained in        step (d) to obtain substantially pure SNS-595 substance.

4.3 Compositions

Compositions are provided that comprise substantially pure SNS-595substance. Compositions are also provided that consist essentially ofsubstantially pure SNS-595 substance. Compositions are also providedthat consist of substantially pure SNS-595 substance.

In some embodiments, compositions are provided wherein the substantiallypure SNS-595 substance comprises about 0 to 0.03% Compound 4

based on total weight of substantially pure SNS-595 substance. In someembodiments, the substantially pure SNS-595 substance comprises about 0to 0.02% or about 0 to 0.01% Compound 4. In some embodiments, thesubstantially pure SNS-595 substance comprises 0 to 0.03%, 0 to 0.02%,or 0 to 0.01% Compound 4.

In some embodiments, substantially pure SNS-595 substances are providedthat consist essentially of SNS-595 and about 0.03 wt % or less, about0.02 wt % or less, or about 0.01 wt % or less Compound 4. In someembodiments, the composition consists essentially of SNS-595 and 0.03 wt% or less, 0.02 wt % or less, or 0.01 wt % or less Compound 4. In someembodiments, the composition consists of SNS-595 and 0.03 wt % or less,0.02 wt % or less, or 0.01 wt % or less Compound 4.

In some embodiments, the substantially pure SNS-595 substance comprisesabout 0 to 0.03%, about 0 to 0.02%, or about 0 to 0.01% Compound 4, atthe time of production. In some embodiments, the composition comprises 0to 0.03%, 0 to 0.02%, or 0 to 0.01% Compound 4, at the time ofproduction.

In some embodiments, substantially pure SNS-595 substances are providedthat consist essentially of SNS-595 and about 0.03 wt % or less, about0.02 wt % or less, or about 0.01 wt % or less Compound 4, at the time ofproduction. In some embodiments, the composition consists essentially ofSNS-595 and 0.03 wt % or less, 0.02 wt % or less, or 0.01 wt % or lessCompound 4, at the time of production. In some embodiments, thecomposition consists of SNS-595 and 0.03 wt % or less, 0.02 wt % orless, or 0.01 wt % or less Compound 4, at the time of production.

In some embodiments, compositions are provided wherein the substantiallypure SNS-595 substance comprises about 0 to 0.04% Compound 5

based on total weight of substantially pure SNS-595 substance. In someembodiments, the substantially pure SNS-595 substance comprises about 0to 0.03%, or about 0 to 0.02% Compound 5. In some embodiments, thesubstantially pure SNS-595 substance comprises 0 to 0.04%, 0 to 0.03%,or 0 to 0.02% Compound 5.

In some embodiments, substantially pure SNS-595 substances are providedthat consist essentially of SNS-595 and about 0.04 wt % or less, about0.03 wt % or less, or about 0.02 wt % or less Compound 5. In someembodiments, the composition consists essentially of SNS-595 and 0.04 wt% or less, 0.03 wt % or less, or 0.02 wt % or less Compound 5. In someembodiments, the composition consists of SNS-595 and 0.04 wt % or less,0.03 wt % or less, or 0.02 wt % or less Compound 5.

In some embodiments, the substantially pure SNS-595 substance comprisesabout 0 to 0.04%, about 0 to 0.03%, or about 0 to 0.02% Compound 5, atthe time of production. In some embodiments, the composition comprises 0to 0.04%, 0 to 0.03%, or 0 to 0.02% Compound 5, at the time ofproduction.

In some embodiments, the substantially pure SNS-595 substance consistsessentially of SNS-595 and about 0.04 wt % or less, about 0.03 wt % orless, or about 0.02 wt % or less Compound 5, at the time of production.In some embodiments, the composition consists essentially of SNS-595 and0.04 wt % or less, 0.03 wt % or less, or 0.02 wt % or less Compound 5,at the time of production. In some embodiments, the composition consistsof SNS-595 and 0.04 wt % or less, 0.03 wt % or less, or 0.02 wt % orless Compound 5, at the time of production.

In some embodiments, substantially pure SNS-595 substances are providedwherein the substantially pure SNS-595 substance comprises about 0 to0.07% total Compound 4 and Compound 5 combined based on total weight ofthe compositions. In some embodiments, the composition comprises about 0to 0.05% total Compound 4 and Compound 5 combined. In some embodiments,the substantially composition comprises about 0 to 0.03% total Compound4 and Compound 5 combined. In some embodiments, the compositioncomprises 0 to 0.07%, 0 to 0.05%, or 0 to 0.03% total Compound 4 andCompound 5 combined.

In some embodiments, substantially pure SNS-595 substances are providedthat consist essentially of SNS-595 and about 0.07 wt % or less, about0.05 wt % or less, or about 0.03 wt % or less total Compound 4 andCompound 5 combined. In some embodiments, the composition consistsessentially of SNS-595 and 0.07 wt % or less, 0.05 wt % or less, or 0.03wt % or less total Compound 4 and Compound 5 combined. In someembodiments, the composition consists of SNS-595 and 0.07 wt % or less,0.05 wt % or less, or 0.03 wt % or less total Compound 4 and Compound 5combined.

In some embodiments, the substantially pure SNS-595 substance comprisesabout 0 to 0.07%, about 0 to 0.05%, or about 0 to 0.03% total Compound 4and Compound 5 combined, at the time of production. In some embodiments,the composition comprises 0 to 0.07%, 0 to 0.05%, or 0 to 0.03% totalCompound 4 or Compound 5 combined, at the time of production.

In some embodiments, substantially pure SNS-595 substances are providedthat consist essentially of SNS-595 and about 0.07 wt % or less, about0.05 wt % or less, or about 0.03 wt % or less total Compound 4 andCompound 5 combined, at the time of production. In some embodiments, thecomposition consists essentially of SNS-595 and 0.07 wt % or less, 0.05wt % or less, or 0.03 wt % or less total Compound 4 and Compound 5combined, at the time of production. In some embodiments, thecomposition consists of SNS-595 and 0.07 wt % or less, 0.05 wt % orless, or 0.03 wt % or less total Compound 4 and Compound 5 combined, atthe time of production.

The presence of Compound 5 in a solution comprising SNS-595 and watercan result in sub-visible microscopic particles. The presence ofmicroscopic or sub-visible particles can be determined by any techniquedeemed suitable by one of ordinary skill in the art. For instance, thenumber of particles can be determined by the obscuration methodspecified in USP-NF General Chapter 788, which is incorporated herein byreference in its entirety and described below. Alternatively, flowimaging techniques (such as that available from Brightwell Technologies,Inc. may be used to determine the particulate matter content of acomposition.

In some embodiments, compositions are provided wherein about 100 mg ofsubstantially pure SNS-595 substance is present for every 10 mL of thecomposition; and wherein the composition has not more than 80, not morethan 70, not more than 60, not more than 50, not more than 40, not morethan 30, not more than 20, not more than 15, not more than 10, or notmore than 5 particles≧25 microns per 10 mL of the composition.

In some embodiments, compositions are provided wherein about 100 mg ofSNS-595 is present for every 10 mL of the composition; and wherein thecomposition has not more than 3000, not more than 2500, not more than2000, not more than 1500, not more than 1000, not more than 800, notmore than 645, not more than 600, not more than 300, or not more than100 particles≧10 microns per 10 mL of the composition.

In some embodiments, compositions are provided that comprise SNS-595 and0.3 mg or less, 0.2 mg or less, or 0.1 mg or less Compound 4 per gram ofSNS-595. In some embodiments, the compositions are aqueous solutions ofa substantially pure SNS-595 substance, e.g., 3 mg or less Compound 4per 10 gm of the substantially pure SNS-595 substance per 100 mLsolution. In some embodiments, the compositions further comprisesorbitol. In some embodiments, the sorbitol is present in an amountproviding a 4.5% aqueous solution of sorbitol. In some embodiments, thecompositions further comprise methanesulfonic acid. In some embodiments,the methanesulfonic acid is present in an amount to provide the solutiona pH of 2.5.

In some embodiments the composition is an aqueous solution consistingessentially of

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595 and Compound 4, wherein the substantially pure SNS-595        substance contains 0.3 mg or less Compound 4 per gram;    -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution. Also provided are products        comprising a container containing an aliquot of such solution,        e.g., 10 mL of such solution.

A composition comprising SNS-595 and 0.4 mg or less, 0.3 mg or less, or0.2 mg or less of Compound 5 per gram of SNS-595. In some embodiments,the compositions are aqueous solutions of a substantially pure SNS-595substance, e.g., 0.4 mg or less of Compound 5 per 10 gm of thesubstantially pure SNS-595 substance per 100 mL solution. In someembodiments, the compositions further comprise sorbitol. In someembodiments, the sorbitol is present in an amount providing a 4.5%aqueous solution of sorbitol. In some embodiments, the compositionsfurther comprise methanesulfonic acid. In some embodiments, themethanesulfonic acid is present in an amount to provide the solution apH of 2.5.

In some embodiments the composition is an aqueous solution consistingessentially of:

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595 and Compound 5, wherein the substantially pure SNS-595        substance contains 0.4 mg or less Compound 5 per gram;    -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution. Also provided are products        comprising a container containing an aliquot of such solution,        e.g., 10 mL of such solution.

A composition comprising SNS-595 and 0.7 mg or less total Compound 4 andCompound 5 per gram of SNS-595. A composition comprising SNS-595 and 0.4mg or less, 0.3 mg or less, or 0.2 mg or less Compound 5 per gram ofSNS-595. In some embodiments, the compositions are aqueous solutions ofa substantially pure SNS-595 substance, e.g., 4 mg or less of Compound 5per 10 gm of the substantially pure SNS-595 substance per 100 mLsolution. In some embodiments, the compositions further comprisesorbitol. In some embodiments, the sorbitol is present in an amountproviding a 4.5% aqueous solution of sorbitol. In some embodiments, thecompositions further comprise methanesulfonic acid. In some embodiments,the methanesulfonic acid is present in an amount to provide the solutiona pH of 2.5.

In some embodiments the composition is an aqueous solution consistingessentially of:

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595, Compound 4, and Compound 5, wherein the substantially        pure SNS-595 substance contains 0.7 mg or less total Compound 4        and Compound 5 combined per gram;    -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution. Also provided are products        comprising a container containing an aliquot of such solution,        e.g., 10 mL of such solution.

Further, the presence of Compound 4 in a solution comprising SNS-595 andwater can result in the formation of visible particles. Without beinglimited to any theory, the visible particles can comprise Compound 6

which can be derived from Compound 4. Without being limited to anytheory, Compound 4, when exposed to formaldehyde, can react to formCompound 6. Indeed, when Compound 4 is treated with formaldehyde, theformation of Compound 6 has been observed.

In some embodiments, processes are provided that, when compared toprocesses known in the art, produce substantially pure SNS-595 substanceincluding lower amounts of Compounds 4 and 5, and, thus, lower amountsof particles, e.g., Compound 6, when such substantially pure SNS-595substance is provided in an aqueous solution.

In some embodiments, processes are provided that, when compared toprocesses known in the art, produce substantially pure SNS-595 substanceincluding lower amounts of compound 7

In some embodiments, compositions comprising substantially pure SNS-595substance are provided, wherein the compositions are essentially free ofvisible particles and are stable over time. In some embodiments, thecomposition is stable for 1, 2, 4, 6, 8, 10, 12, 15, 20, or 25 day(s).In some embodiments, the composition is stable for 1, 3, 6, 9, 12, 18,24, 36, or 42 month(s). In some embodiments, the composition is stablestarting from the time of production. In some embodiments, thecomposition is stable when contacted with formaldehyde. In someembodiments, the composition o is stable when contacted with a compoundcapable of transforming Compound 4

to Compound 6

As discussed, visible particles may have, in some embodiments, anaverage diameter of at least 50 μm, at least 75 μm, at least 100 μm, atleast 150 μm, or at least 200 μm. In some embodiments the visibleparticles have an average diameter of about 50-500 μm, about 50-300 μm,about 100-500 μm, or about 100-300 μm.

The crystallinity and crystalline habit may be determined using methodsknown to those of ordinary skill in the art. For example, crystallinityand crystalline habit may be assessed by polarized light microscopy. Insome embodiments, the visible particles are crystalline powders. In someembodiments, the crystalline habit of the visible particles is that ofplates about 45 μm to about 150 μm, or about 50 μM to about 100 μm.

The detection of visible particles in a composition can be determined byany technique deemed suitable by one of ordinary skill in the art. Forinstance, visible particles may be detected by the method specified inEuropean Pharmacopeia 5.0, section 2.9.20, which is incorporated hereinby referenced in its entirety. Certain exemplary techniques aredescribed in greater detail below.

In some embodiments, visible particle presence is determined at anillumination intensity between about 2000 and 3750 lux.

In some embodiments, the visible particles comprise compound 6.

In some embodiments, the visible particles comprise compound 7.

In some embodiments, processes are provided that, when compared toprocesses known in the art, produce substantially pure SNS-595 substanceincluding lower amounts of sub-visible particles as detected by theLight Obscuration Particle Count Test as described in the U.S.Pharmacopoeia, <788> Particulate Matter in Injections, the entirety ofwhich is incorporated herein by reference. In some embodiments, thesub-visible particles comprise one or more of compounds 5, 6, and/or 7.

In some embodiments, provided herein are compositions comprisingsubstantially pure SNS-595 substance, wherein the compositions containnot more than 6000 sub-visible particles ≧10 μm per vial, not more than3000 sub-visible particles ≧10 μm per vial, or not more than 1000sub-visible particles ≧10 μm per vial as measured by light obscuration.In some embodiments, provided herein are compositions comprisingsubstantially pure SNS-595 substance, wherein the compositions containnot more than 600 sub-visible particles particles ≧25 μM per vial, notmore than 300 sub-visible particles particles ≧25 μm per vial, or notmore than 100 sub-visible particles ≧25 μm per vial by lightobscuration. In some embodiments, provided herein are compositionscomprising substantially pure SNS-595 substance, wherein thecompositions contain not more than 3000 sub-visible particles ≧10 μm pervial, not more than 1500 sub-visible particles ≧10 μm per vial, or notmore than 300 sub-visible particles ≧10 μm per vial as measured bymicroscopic evaluation. In some embodiments, provided herein arecompositions comprising substantially pure SNS-595 substance, whereinthe compositions contain not more than 300 sub-visible particles ≧25 μmper vial, not more than 150 sub-visible particles ≧25 μm per vial, ornot more than 30 sub-visible particles ≧25 μm per vial as measured bymicroscopic evaluation.

In some embodiments, the composition comprises sorbitol. In furtherembodiments, the composition comprises methanesulfonic acid. In furtherembodiments, the sorbitol is present in an amount providing a 4.5%aqueous solution of sorbitol. In some embodiments, the methanesulfonicacid is present in an amount to provide a pH of 2.5. In someembodiments, the composition is stable.

In some embodiments, the composition consists essentially of 100 mgsubstantially pure SNS-595 substance, 450 mg of D-sorbitol, water, andmethanesulfonic acid, wherein the methanesulfonic acid is present inamount to provide a pH of 2.5, and wherein the water is present in anamount to provide a total composition volume of 10 mL. In someembodiments, the composition is stable.

In some embodiments, the composition consists essentially of water,substantially pure SNS-595 substance, sorbitol, and methanesulfonicacid. In some embodiments, the composition is stable.

In some embodiments, compositions consisting essentially of SNS-595 and0.03 wt % or less Compound 4 are provided.

In some embodiments, compositions consisting essentially of SNS-595 and0.04 wt % or less Compound 5 are provided.

In some embodiments, compositions consisting essentially of SNS-595 and0.07 wt % or less total Compound 4 and Compound 5 are provided.

In some embodiments, compositions comprising SNS-595 and 0.3 mg or lessCompound 4 per gram of SNS-595 are provided.

In some embodiments, compositions comprising SNS-595 and 0.4 mg or lessCompound 5 per gram of SNS-595 are provided.

In some embodiments, compositions comprising SNS-595 and 0.7 mg or lesstotal Compound 4 and Compound 5 per gram of SNS-595 are provided.

In some embodiments, compositions consisting essentially of:

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595 and Compound 4 wherein the substantially pure SNS-595        substance contains 3 mg or less Compound 4 per gram

-   -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution are provided. In some embodiments, a        product comprising a container containing 10 mL of such solution        is provided.

In some embodiments, an aqueous solution consisting essentially of:

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595 and Compound 5

-   -   wherein the substantially pure SNS-595 substance contains 4 mg        or less Compound 5 per gram;    -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution is provided. In some embodiments, a        product comprising a container containing 10 mL of such solution        is provided.

In some embodiments, an aqueous solution consisting essentially of:

-   -   (a) 10 g of a substantially pure SNS-595 substance consisting of        SNS-595, Compound 4 and Compound 5

-   -   wherein the substantially pure SNS-595 substance contains 7 mg        or less total Compound 4 and Compound 5 combined per gram;    -   (b) 4.5 g of sorbitol: and    -   (c) sufficient methanesulfonic acid to provide a pH of 2.5;        per 100 mL of the solution is provided. In some embodiments, a        product comprising a container containing 10 mL of such solution        is provided.

Also provided herein is a compound of formula 6

or a pharmaceutically acceptable salt thereof.

Also provided herein is a compound of formula 7

or a pharmaceutically acceptable salt thereof

6. EXAMPLES

Certain embodiments of the claimed subject matter are illustrated by thefollowing non-limiting examples.

Example 1 Reduction of Compound 4 by Reprocessing

As noted, the reaction of Compound 1 with Compound 2 can result in theformation of a mixture of Compound 3 and residual Compound 1. Subsequenttreatment of this mixture with aqueous base, i.e., saponificationconditions with, for example, aqueous sodium hydroxide, can result inthe formation of a mixture of SNS-595, Compound 4, and Compound 5.However, when this mixture is again subjected to the saponificationconditions, i.e., reprocessed, the resulting product can contain SNS-595with lower amounts of Compound 4, as seen above in Scheme 6.

Experiments were performed to assess the effect of such reprocessing ofSNS-595 substance on the residual levels of Compound 4. The results ofthese experiments are presented in Table 1.

TABLE 1 Reduction of Compound 4 by reprocessing Compound CompoundCompound 4 spiked 4 after NaOH 4 after (actual) treatment reprocessingExperiment Entry Scale wt % area % area % (wt %) 1 1 1 g 1 0.70 0.06 2None 0.06 <0.01 3 <0.01 <0.01 2 4 1 g 1 0.89 0.06 5 None 0.06 ND 3 6 25g 0.3 NA  0.01 (0.033) 4 7 25 g 0.2 NA 0.005 (0.015) 5 8 25 g 0.1 NA0.002 (0.003) 6 9 40 g 0.02¹ NA (0.004) 7 10 200 g 0.02¹ NA (0.005) 8 111.49 kg 0.02¹ NA (<0.005) 9 12 1.2 kg 0.02¹ NA (<0.015) ¹SNS-595 with0.02% Compound 4 was used NA = not available; ND = None detected

Experiment 1:

To a solution of sodium hydroxide (0.15 g) in water (5 mL), SNS-595(0.98 g), Compound 4 (0.010 g) and ethanol (EtOH) (1 mL) were added. Themixture was filtered and the HPLC analysis of the filtrate showedCompound 4 as about 0.7% (by area). The pH of the filtrate was adjustedto 6 by slow addition of acetic acid and the solution was heated toprecipitate SNS-595 hydrate. The slurry was cooled and filtered to giveSNS-595 hydrate. HPLC analysis of the solid showed it to have 0.06% (byarea) of Compound 4, indicating that a ˜10-fold reduction in Compound 4had occurred. The solid was re-subjected to reprocessing using aqueoussodium hydroxide (0.18 g in 6 mL water) and EtOH (1 mL), followed by pHadjustment with aqueous acetic acid. After heating, the solid wasfiltered to give SNS-595 hydrate having less than 0.01% Compound 4,demonstrating at least a further six-fold reduction in the amount of thecontaminant.

Experiment 2: SNS-595 (˜1 g) was spiked with 1% Compound 4. HPLCanalysis of this spiked sample following reprocessing conditions, i.e.,treatment with NaOH, showed 0.89% Compound 4 by area. This material wasthen pH-adjusted and the solid isolated as above. HPLC analysis showed0.06% Compound 4 by area, indicating that a ˜10-fold reduction inCompound 4 occurred, consistent with the first experiment above. Thismaterial, when again subjected to reprocessing conditions, provided aSNS-595 substance with no detectable Compound 4, demonstrating at leasta six-fold reduction in the amount of that contaminant.

Scalability Experiments:

Experiments 3-9 were performed to evaluate the scalability of thisprocess. The procedure for Experiment 8 is provided as an exemplaryprocedure. Results for Experiments 3-9 are summarized below and on Table1.

Exemplary Procedure for Large Scale Reprocessing (Experiment 8):

To a solution of NaOH (0.2 kg) in water (4.8 kg), SNS-595 (1.49 kg)having Compound 4 (˜0.24% by weight) was added. To the mixture, EtOH(0.13 kg) was added. The solution was filtered and the pH of thefiltrate was adjusted to 7.3-7.7 by slow addition of aqueous aceticacid. The mixture was heated to 55-65° C., cooled, and filtered. Thefilter cake was washed with water, EtOH and dried under vacuum to give aSNS-595 hydrate (1.36 kg) having 0.013% (by weight) Compound 4. Thehydrate (1.36 kg) was dehydrated by slurrying in EtOH (23 kg) at 67-78°C. After cooling, the mixture was filtered, washed with EtOH and driedat 65-75° C. under vacuum to give a SNS-595 substance (1.0 kg) havingless than 0.005% Compound 4.

Summary of Scalability Experiments:

In Experiment 3, 25 g of SNS-595 having 0.3% Compound 4 was subjected toreprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showed0.01% Compound 4 by area, indicating that a ˜30 fold reduction inCompound 4 had occurred.

In Experiment 4, 25 g of SNS-595 having 0.2% Compound 4 was subjected toreprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showed0.005% Compound 4 by area, indicating that a ˜40 fold reduction inCompound 4 had occurred.

In Experiment 5, 25 g of SNS-595 having 0.1% Compound 4 was subjected toreprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showed0.002% Compound 4 by area, indicating that a ˜50 fold reduction inCompound 4 had occurred.

In Experiment 6, 40 g of SNS-595 having 0.02% Compound 4 was subjectedto reprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showed0.004% Compound 4 by area, indicating that a ˜5-fold reduction inCompound 4 had occurred.

In Experiment 7, 200 g of SNS-595 having 0.02% Compound 4 was subjectedto reprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showedless than 0.005% Compound 4 by area, indicating that a ˜4-fold reductionin Compound 4 had occurred.

In Experiment 8, 1.49 kg of SNS-595 having 0.02% Compound 4 wassubjected to reprocessing conditions, i.e., sodium hydroxide, followedby pH adjustment. HPLC analysis showed <0.005% Compound 4 by area,indicating that a ˜4 fold reduction in Compound 4 occurred.

In Experiment 9, 1.2 kg of SNS-595 having 0.02% Compound 4 was subjectedto reprocessing conditions, i.e., sodium hydroxide, followed by pHadjustment and dehydration. HPLC analysis of resulting SNS-595 showed<0.015% Compound 4 by area, indicating that a ˜2-fold reduction inCompound 4 had occurred.

Example 2 Reduction of Amount of Compound 6

As discussed, the presence of Compound 4 in a SNS-595 solution may beattended by the formation of visible particles, which, without beinglimited to any theory, comprise Compound 6. Experiments were performedto assess the effect of reducing Compound 4 levels on the formation ofCompound 6 over time.

Four different sample drug product solutions were prepared from the drugsubstances having varying levels of Compound 4 outlined in Table 2,below. Sample 1 used the reprocessed product obtained from Experiment 6(described in Example 1) and had <0.005% Compound 4. Whenever externaladdition of Compound 4 was made, Compound 4 was first dissolved in a 5mM aqueous NaOH solution. A total of 2% of the aqueous NaOH solution wasadded to each of the drug product Samples for consistency purposes.

The various drug product Samples were filtered twice through a 0.22 μmPVDF SteriCup filters. These pre-autoclaved, filtered drug productSamples were analyzed for their Compound 4 content six days followingpreparation and storage at room temperature (RT) shielded from light.FIG. 1 and Table 2 show that the amount of Compound 6 in the drugproduct correlated directly with the level of Compound 4 in the SNS-595drug substance.

The filtered drug product Samples were then filled in 30 mL Schott glassvials and stoppered. Vial filling and stoppering were performed in alaminar flow hood. Vials and stoppers used in this study were rinsedwith filtered water and allowed to dry in the laminar flow hood beforeuse. Vials were not depyrogenated and stoppers were not sterilizedbefore use. Filled and stoppered vials were crimp-sealed, autoclaved,and stored upright and shielded from light at 40° C./75% relativehumidity (RH). After 65 days of storage, the vials were inspected forthe presence of visible particles and analyzed by HPLC for Compound 6content. Analysis of the Samples having less than 0.01% Compound 4(entry 1) showed that the resulting drug product had no visibleparticles and no detectable amount of Compound 6 after 65 days.Experiments with Samples having ≧0.02% Compound 4 showed significantformation of Compound 6 and visible particles during the same timeperiod (Samples 2-4). The amount of Compound 6 after 65 days correlateddirectly with the initial amount of Compound 4 in the SNS-595 substance.

TABLE 2 Effect of Compound 4 on Compound 6 levels Nominal Compound 4 inCompound 6 Compound 6 Drag Substance (μg/vial) (μg/vial) Sample (% w/w)After ~6 days After 65 days 1 <0.01 0.0 0.0 2 0.02 0.6 7-9 3 0.04 ~214-19 4 0.20 ~9 16-24

Example 3 Reduction of Compounds 4 and 5 by Via Wet DIPEA

Experiments were performed to assess the effect of water on the reactionbetween Compound 1 and Compound 2 in the presence of DIPEA. Experimentswere also performed to compare the effect of adding water during thebeginning of the reaction with adding water at the end of the reaction.

When Compound 1 and Compound 2 were reacted at 40-45° C. in the absenceof water, analysis of the product showed between 0.26% and 0.31%residual Compound 1 by area.

In a second series of experiments, Compound 1 and Compound 2 werereacted at RT for 12 hr, and subsequently heated to 40-45° C. for 3-5hr. In these experiments, water was added at the beginning of thereaction. Analysis of the product showed between 0.01% and 0.08%residual Compound 1 by area.

In a third series of experiments, Compound 1 and Compound 2 were reactedat RT for 12 hr, and subsequently heated to 45° C. for 3 hr. In theseexperiments, water was added at the end of the reaction. Analysis of theproduct showed 0.03% to 0.09% residual Compound 1 by area.

The experiments above are summarized in the table below.

TABLE 3 Effect of water in SNS-595 synthesis Residual Water Compound 1Rxn Condition (volume) Scale (area %)* Comments DIPEA, 40-45° C. 50 g0.26 0 0.31 No water DIPEA, RT, 12 hr, 0.5 1 g 0.05 Water added thenheated to 40- 10 g 0.08 at the 45° C., 3-5 hr 50 g 0.08 beginning 100 g0.05 1 kg 0.01 DIPEA, RT, 12 hr, 0.5 10 g 0.03 Water added then heatedto 40- 10 g 0.05 after 12 hr 45° C., 3 hr 50 g 0.09 at RT 100 g 0.07*Analyzed at 275 nm

The following is an exemplary procedure for the experiments described inthe Table 3 above.

To a slurry of Compound 2 (1.55 kg) in acetonitrile (ACN; 10 L), DIPEA(4 L) and water (0.5 L) were added. To the solution, Compound 1 (1 kg)and ACN (1 L) were added and the reaction was stirred for about 12 hr atRT. The reaction mixture was then heated to about 45° C. for about 2-6hr. After cooling, the product was filtered, washed with ACN (4 L) anddried under vacuum to give Compound 3 (1.1 kg). HPLC analysis showedthis material to contain <0.1% Compound 1.

Experiments were carried out to determine the impact of residualCompound 1 levels on the Compound 4 levels resulting during themanufacture of SNS-595. These experiments demonstrated a correlationbetween Compound 1 levels in Compound 3 and Compound 4 levels in theresulting SNS-595 substance. Experiments at laboratory scale alsodemonstrated that using Compound 3 substance having <0.1% Compound 1provided a SNS-595 substance with <0.01% Compound 4. The results ofthese experiments are summarized below in Table 4 and FIG. 2.

TABLE 4 Impact of residual Compound 1 in Compound 3 on Compound 4 levelsin SNS-595 Compound 1 Compound 4 Entry Scale Area % (wt %) 1 0.15 kg0.50 0.04 2 0.75 kg 0.60 0.05 3 2.1 kg 0.64 0.04 4 5 kg 0.24 0.02 5 10 g0.08 0.007 6 50 g 0.09 0.008 7 1 kg 0.01 <0.005

The following is an exemplary procedure for the experiments described inTable 4 above.

Compound 3 (0.9 kg) (having <0.1% Compound 1) was added to a solution ofsodium hydroxide (0.14 kg) in water (3.3 kg) and EtOH (0.16 L). Themixture was stirred for about 12 hr and filtered to remove insolublematerials. The pH of the filtrate was adjusted to 7.3-7.7 with aqueousacetic acid. The resulting mixture was heated to about 60° C. for 2-4hr, cooled and filtered. The filter cake was washed with water, EtOH anddried under vacuum. The resulting SNS-595 hydrate was slurried in EtOH(20 L) at about 70° C. for about 4 hr, cooled and filtered. The filtercake was washed with EtOH and dried at 55-75° C. under vacuum to giveSNS-595 substance (0.66 kg) having less than 0.005% Compound 4 (Table 4,entry 7).

In a similar manner, experiments were carried out to determine theimpact of residual Compound 1 levels on the Compound 5 levels inSNS-595. These experiments demonstrated a correlation between Compound 1levels in Compound 3 and Compound 5 levels in SNS-595. The results ofthese experiments are summarized below in Table 5 and FIG. 3.

TABLE 5 Impact of residual Compound 1 on Compound 5 levels in SNS-595Compound 1 Compound 5 Entry Scale (Area %) (wt %) 1 0.75 kg 0.60 0.106 22.1 kg 0.64 0.077 3 5 kg 0.24 0.028 4 50 g 0.3 0.019 5 50 g 0.2 0.016 650 g 0.1 0.011 7 50 g 0.1 0.011 8 1 kg 0.01 <0.01

Example 4 Pharmaceutical Composition Suitable for Injection orIntravenous Infusion and Determination of Particle Impurity

An illustrative example of a suitable SNS-595 pharmaceutical compositioncomprises: 10 mg of substantially pure SNS-595 drug substance per mL ofaqueous solution of 4.5% sorbitol that is adjusted to pH 2.5 withmethanesulfonic acid. One protocol for making such a solution includesthe following for making a 100 mg/10 mL presentation: 100 mgsubstantially pure SNS-595 substance (prepared following the methodsdescribed herein) and 450 mg D-sorbitol are added to distilled water;the volume is brought up to a volume of 10 mL; and the pH of theresulting solution is adjusted to 2.5 with methanesulfonic acid.

The resulting SNS-595 composition will be essentially free of visibleparticles and stable.

As noted, the presence of particulate matter can be determined using anyconvenient technique. For example, the presence of visible particles canbe determined according to the method described by European Pharmacopeia5.0, Section 2.9.20, the entirety of which is incorporated herein byreference. Specifically, an apparatus having a viewing stationcomprising (1) a matt black panel of appropriate size held in a verticalposition; (2) a non-glare white panel of appropriate size held in avertical position next to the black panel; and (3) an adjustablelampholder fitted with a suitable, shaded, white-light source and with asuitable light diffuser (e.g., a viewing illuminator containing two 13 Wfluorescent tubes, each 525 mm in length) is used. The intensity ofillumination at the viewing point is maintained between 2000 lux and3750 lux. Adherent labels are removed from the container. The outside ofthe container is washed and dried on the outside. The container isgently swirled or inverted while ensuring that air bubbles are notintroduced, and the container is observed for about 5 seconds in frontof the white panel to determine the presence of visible particles. Thecontainer is then observed for about 5 seconds in front of the blackpanel to determine the presence of visible particles. If visibleparticles are detected, from viewing in front of either panel, thecorresponding container is rejected.

Further, the light obscuration particle count test described in USP-NFGeneral Chapter 788 may be used, which is incorporated herein byreference in its entirety. Specifically, a suitable apparatus based onthe principle of light blockage which allows an automatic determinationof the size of particles and the number of particles according to sizeis used. The apparatus is calibrated using dispersions of sphericalparticles of known sizes between 10 μm and 25 μm, USP Particle CountReference Standard. These standard particles are dispersed inparticle-free water. Care is taken to avoid aggregation of particlesduring dispersion.

The test is carried out under conditions limiting particulate matter,for example in a laminar-flow cabinet. The glassware and filtrationequipment used is very carefully washed, except for the membranefilters, with a warm detergent solution and rinsed with abundant amountsof water to remove all traces of detergent. Immediately before use, theequipment is rinsed from top to bottom, outside and then inside, withparticle-free water.

Care is taken not to introduce air bubbles into the preparation to beexamined, especially when fractions of the preparation are beingtransferred to the container in which the determination is to be carriedout. To check that the environment is suitable for the test, that theglassware is properly cleaned and that the water to be used isparticle-free, the following test is carried out: determine theparticulate matter in 5 samples of particle-free water, each of 5 mL,according to the method described below. If the number of particles of10 μm or greater size exceeds 25 for the combined 25 mL, the precautionstaken for the test are not sufficient. The preparatory steps arerepeated until the environment, glassware and water are suitable for thetest.

The contents of the sample are mixed by slowly inverting the container20 times successively. If necessary, the sealing closure is cautiouslyremoved. The outer surfaces of the container opening are cleaned using ajet of particle-free water and the closure is removed, avoiding anycontamination of the contents. Gas bubbles are eliminated by appropriatemeasures such as allowing the sample to stand for 2 min or bysonication.

For small-volume parenteral products less than 25 mL in volume, thecontents of 10 or more units are combined in a cleaned container toobtain a volume of not less than 25 mL; the test solution may beprepared by mixing the contents of a suitable number of vials anddiluting to 25 mL with particle-free water or with an appropriateparticle-free solvent when particle-free water is not suitable.Parenteral products having a volume of 25 mL or more may be testedindividually.

The number of test specimens is such to afford a statistically soundassessment. For large-volume parenterals or for small-volume parenteralshaving a volume of 25 mL or more, fewer than 10 units may be tested,based on an appropriate sampling plan.

Four samples are removed from the specimen, each of not less than 5 mL,and the particles having diameters equal to or greater than each of 10μm and 25 μm are counted. The result obtained for the first sample isdisregarded, and the mean number of particles for the specimen iscalculated.

Example 5 Synthesis of Compound 4

Compound 1 (100 g; 1.0 eq) was added to a solution of 25 g of LiOH—H₂O(0.25 eq) in 625 mL water and 125 mL EtOH. The resulting slurry wasmixed was stirred at 25-30° C. overnight. The reaction mixture was thenfiltered and washed with 500 mL water and 500 mL EtOH. The solids werethen dried at 50° C. overnight in vacuo to afford Compound 4. ¹H NMR(400 MHz, DMSO-d₆): δ 9.95 (s, 1H), 8.5 (d, J=8.4 Hz, 1H), 7.95 (d, J=4Hz, 1H), 7.88 (d, J=3.2 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H); ¹³C NMR (125MHz, DMSO-d₆): 193.0, 171.2, 162.0, 155.6, 153.7, 152.0, 140.9, 137.9,124.4, 119.9, 110.9, 106.4; MS: m/z 310, 308 (M+H)⁺.

Example 6 Synthesis of Compound 6

Compound 4 (10 g) was taken in 500 mL acetic acid. The resulting slurrywas stirred for 15 minutes (min) at RT under nitrogen atmosphere. Theslurry was then added to a 10 L 3-necked round bottom flask containing4.5 L of 37% aqueous formaldehyde solution. The resulting slurry wasthen stirred at 60-62° C. overnight and cooled to 25-30° C. The reactionmixture was then filtered, and the resulting white powder was driedunder vacuum at 25-30° C. overnight. The solid was re-suspended in 150mL acetic acid and stirred for 3 days at RT. The resulting slurry wasthen filtered, and the solids were dried in a vacuum overnight to affordCompound 6. ¹H NMR (400 MHz, DMSO-d₆): δ 8.38 (d, J=8.8 Hz, 2H), 7.97(d, J=3.6 Hz, 2H), 7.89 (d, J=3.6 Hz, 2H), 7.46 (d, J=8 Hz, 2H), 3.89(s, 2H); MS: m/z 571, 573, 575 (M+H)⁺.

Compound 6 was analyzed by polarized light microscopy to assess itscrystallinity and crystalline habit. Compound 6 appears to be acrystalline powder under polarized light microscope as it exhibitsstrong birefringence. The crystalline habit is that of plates 50-100 μm,as isolated from acetic acid.

Example 7 Preparation of Substantially Pure SNS-595 Substance from2,6-Dichloronicotinic Acid and N-Boc-3-pyrroline Preparation of Compound1 from 2,6-dichloronicotinic acid

A solution of carbonyldiimidazole (CDI) (16.4 kg) in tetrahydrofuran(THF) was added to a slurry of 2,6-dichloronicotinic acid (Compound A)(16 kg) in THF. After about 2 hr, ethyl potassium malonate(EtO₂CCH₂CO₂K) (19.4 kg), triethylamine (25.9 kg) and magnesium chloride(11.9 kg) were added and the reaction stirred for about 24 hr. Thereaction mixture was quenched with dilute HCl and extracted with ethylacetate. The organic layer was concentrated, washed with a mixture ofaqueous NaCl and NaHCO₃. The organic layer was diluted withmethylcyclohexane and dried by vacuum distillation. The solution wastreated with triethylorthoformate (17.1 kg) and acetic anhydride (59 kg)at about 90 to 110° C. After the reaction was judged to be complete, theexcess acetic anhydride was removed by distillations withmethylcyclohexane. The crude product was treated with a solution of2-aminothiazole (8.2 kg) in THF. After about 2 hr, the reaction mixturewas treated with potassium carbonate (13.6 kg) and the mixture stirredfor about 6 hr. The product was precipitated by the addition of water,isolated by filtration, washed with ACN-water, ACN, and dried to giveCompound 1 (13.1 kg).

Preparation of Compound 2 N-Boc-3-pyrroline

(±)-3-Bromo-4-hydroxy-pyrrolidine-1-carboxylic acid, tert-butyl ester(2). (Tetrahedron Asymmetry, 12 (2002) 2989-2997)

N-Boc-3-pyrroline B (296 g, 1.75 moles) was added to a slurry of1,3-dibromo-5,5-dimethylhydantoin (270 g, 0.94 moles) in acetonitrile(ACN, 1800 mL) and water (296 mL), while maintaining the temperature ofthe vessel at 0 to 10° C. After the addition, the reaction mixture waswarmed to RT and stirred until the reaction was judged to be complete(TLC or HPLC). The reaction was quenched by the addition of 5% aqueoussodium thiosulfate solution (600 mL) and the product was extracted withdichloromethane (2×750 mL). The combined organic layer was washed withwater (300 mL) and brine (200 mL). The organic layer was dried overanhydrous Na₂SO₄ (75 g) and concentrated under reduced pressure to giveCompound B (450 g) which was directly used in the next step.

6-Oxa-3-aza-bicyclo[3.1.0]hexane-3-carboxylic acid, tert-butyl ester(Compound D). An aqueous solution of sodium hydroxide (NaOH, 1.55 L, 2N)was added to Compound C (450 g, 1.69 moles) and the reaction was stirredbetween for 2 hr at about RT. The product was extracted withdichloromethane (2×1.25 L) and the combined organic layer was washedwith water (2×750 mL) to neutral pH and then dried over anhydrousNa₂SO₄. Evaporation under reduced pressure gave the epoxide D (291.0 g).

(±)-3-Hydroxy-4-methylamino-pyrrolidine-1-carboxylic acid, tert-butylester (Compound E). Aqueous methylamine solution (40% solution, 812 mL,3.8 mol) was added to the epoxide D (140 g, 0.65 mol) at RT and thereaction was stirred until complete. The excess methylamine was removedby distillation under reduced pressure. To the residue obtained,diisopropyl ether (800 mL) was added and the mixture stirred for about30 min. The solid was filtered, washed with diisopropyl ether (200 mL),then dried to give Compound E (135 g).

(±)-3-Hydroxy-4-methylamino-pyrrolidine-1-carboxylic acid, tert-butylester (Compound E), from Compound C.

Ten grams (10 g) of bromohydrin (Compound C) was treated with 40%aqueous methylamine (50 mL) and sodium bicarbonate (3.1 g) at RT to giveCompound E (8.5 g).

Resolution of (±)-3-Hydroxy-4-methylamino-pyrrolidine-1-carboxylic acid,tert-butyl ester, using L-(−)-malic acid. The aminoalcohol (Compound E)(100 g, 0.46 moles) was dissolved in a mixture of acetone (600 mL) andwater (13 mL) at RT. The reaction mixture was heated to about 40° C. andL-(−)-malic acid (62 g, 0.48 moles) was added. The mixture was heated toabout 50 to 55° C. to form a clear solution and then gradually cooled toRT and then to 5 to 10° C. The crystals formed were filtered, washedwith acetone (2×70 mL), and dried under reduced pressure to give themalate salt F (60 g, 37%), with purity by chiral HPLC ratio of S to Renantiomers (S:R)=100:0.

A small sample was analyzed for enantiomeric purity by conversion toCompound G and analyzing the resulting Compound G by chiral HPLC(Chiracel OD-H SC\522; mobile phase: hexane:IPA 95:5; 1 mL/min). Theretention time for the S-enantiomer is 7.725 min.

Resolution of (±)-3-Hydroxy-4-methylamino-pyrrolidine-1-carboxylic acid,tert-butyl ester, using (L)-(−)-pyroglutamic acid. Resolution ofCompound E (10 g) with (L)-(−)-pyroglutamic acid (3.58 g) in acetone(120 mL) and water (4 mL) gave the pyroglutamate salt (5.7 g).Crystallization from acetone-water gave 4.2 g of the PGA salt with 94:6ratio of diastereomers. An additional recrystallization fromacetone-water gave the diastereomerically pure PGA salt (2.3 g, >99%de).

Preparation of3-(tert-Butoxycarbonyl-methyl-amino)-4-hydroxy-pyrrolidine-1-carboxylicacid, tert-butyl ester (Compound G) from L-(−)-malic acid salt (CompoundF)

To a mixture of Compound F (57 g, 0.16 moles) in methanol (MeOH, 220mL), K₂CO₃ (68.0 g, 0.49 moles) was added at RT. Boc anhydride (40 g,0.18 moles) was added dropwise to the reaction mixture over about 1 hrand the reaction mixture was stirred until the reaction was complete(about 2 hr). Methanol was distilled off under reduced pressure at about55 to 60° C., water (150 mL) was added to the reaction mixture and theproduct was extracted with methyl tert-butyl ether (MTBE, 2×150 mL). Thecombined organic layer was washed with water (200 mL) and brine (100mL), and then dried over anhydrous Na₂SO₄. Concentration under reducedpressure gave Compound G as a white solid (52 g).

3-(tert-Butoxycarbonyl-methyl-amino)-4-methoxy-pyrrolidine-1-carboxylicacid, tert-butyl ester (Compound H).

A suspension of Compound G (52 g, 0.16 mol) in THF (150 mL) was stirredat RT for about 30 min and cooled to −10 to −15° C. A solution ofpotassium hexamethyldisilylamide (KHMDS, 40 solution in THF, 144 mL,0.256 mol) was slowly added while controlling the temperature between −5and −15° C. After 15 min, dimethyl sulfate (18.7 mL, 1.20 mol) was addeddropwise to the reaction mixture while maintaining a temperature between−10 and 0° C., and the resulting reaction mixture was then stirred atthis temperature for about 30 min. The reaction mixture was quenched bythe addition of water (100 mL), followed by acetic acid (50 mL). Theproduct was extracted with methyl tert-butyl ether (2×150 mL). Thecombined organic layer was washed with water (100 mL), brine (50 mL) anddried over anhydrous Na₂SO₄. Evaporation under reduced pressure gaveCompound H as an oil (54 g).

(+)-(4-Methoxy-pyrrolidin-3-yl)-methyl-amine (Compound 2), preparedusing toluene-4-sulfonic acid (2:1). To a solution of Compound H (54.0g, 0.163 moles) in THF (180 mL) and MeOH (90 mL), p-toluene sulfonicacid monohydrate (84 g, 0.442 moles) was added and the reaction mixturewas heated to 55-60° C. for about 5 hr, at which time the deprotectionwas complete. After cooling to about 40-45° C., 0.2 g seed crystals ofCompound 2 was added to the reaction mixture resulting in immediatecrystallization. The slurry was maintained at 40-45° C. for about 30minutes and then gradually cooled to 0-5° C. After agitating for 2 hr at0-5° C., solids were filtered, washed with THF (2×50 mL), and dried togive the tosylate salt Compound 2 as a white solid (66 g) with HPLCpurity=98.9%.

The HPLC conditions were as follows: Column: Chiralcel AD H, SC\523;mobile phase: Heptane: IPA (0.5% TFA)=85:15; flow rate: 1.0 mL/min, andruntime: 20 min.

Compound 2 has the retention time of 12.66 min. Enantiomeric excess ofthis material was greater than 99% ee.

Preparation of Substantially Pure SNS-595 Substance Via Reprocessing

To a slurry of Compound 2 (8.0 kg) in ACN at about 5° C., DIPEA (8.7 kg)is added. After about 15 min, Compound 1 (5.0 kg) is added to thereaction mixture. The reaction mixture is heated to about 45° C. forabout 3 hr, cooled and the product filtered. The filter cake is washedwith ACN and dried to give Compound 3.

To a solution of NaOH (0.8 kg) in water (19.5 kg), Compound 3 (5.5 kg)and EtOH (0.5 kg) are added. The reaction mixture is filtered and thefiltrate acidified to pH 7.3-7.7 by the addition of acetic acid. Themixture is then heated to about 55-65° C. for about 2 hr. After coolingto ambient temperature, the reaction mixture is filtered and washed withwater and then with EtOH. The filter cake is dried under vacuum. Thecrude product is slurried in EtOH at about 80° C. After cooling, theproduct is filtered, washed with EtOH and dried to give a SNS-595mixture.

Next, to a solution of NaOH (0.2 kg) in water (4.8 kg), the SNS-595mixture obtained above (1.49 kg) and EtOH (0.13 kg) are added. Thereaction mixture is filtered and the filtrate acidified to pH 7.3-7.7 bythe addition of aqueous acetic acid (prepared from 0.9 kg acetic acidand 2.9 kg water). The mixture is then heated to about 55-65° C. forabout 2 hr. After cooling to ambient temperature, the reaction mixtureis filtered and washed with water and then with EtOH. The filter cake isdried under vacuum. The crude product is slurried in EtOH at about 80°C. After cooling, the product is filtered, washed with EtOH and dried togive a substantially pure SNS-595 substance.

Preparation of Substantially Pure SNS-595 Substance Via WetN,N-diisopropylethylamine

To a slurry of Compound 2 (1.55 kg) in acetonitrile (ACN; 10 L),diisopropylethylamine (DIPEA; 4 L) and water (0.5 L) were added. To thesolution, Compound 1 (1 kg) and acetonitrile (1 L) were added and thereaction was stirred for about 12 hr at RT. The reaction mixture wasthen heated to about 45° C. for about 2-6 hr. After cooling, the productwas filtered, washed with ACN (4 L) and dried under vacuum to giveCompound 3 (1.1 kg). HPLC analysis showed this material to contain <0.1%Compound 1.

To a solution of NaOH (0.135 kg) in water (3.3 L), substantially pureCompound 3 (0.9 kg) and EtOH are added. After hydrolysis was complete,the reaction mixture was filtered and the filtrate acidified to pH7.3-7.7 by the addition of aqueous acetic acid. The mixture was thenheated to about 55-65° C. for about 2 hr. After cooling to ambienttemperature, the reaction mixture was filtered and washed with water andthen with EtOH. The filter cake is dried under vacuum. The crude productwas slurried in EtOH at about 80° C. After cooling, the product wasfiltered, washed with EtOH, and dried to give a substantially pureSNS-595 substance (0.66 kg).

The embodiments of the claimed subject matter described above areintended to be merely exemplary, and those skilled in the art willrecognize, or will be able to ascertain using no more than routineexperimentation, numerous equivalents of specific compounds, materials,and procedures. All such equivalents are considered to be within thescope of the claimed subject matter and are encompassed by the appendedclaims.

1. A process for preparing a substantially pure SNS-595 substance,comprising: (a) reacting Compound 3

with a first aqueous base followed by neutralizing to obtain a primarySNS-595 hydrate; (b) dehydrating the primary SNS-595 hydrate from step(a) and reacting the dehydrated product with a second aqueous basefollowed by neutralizing to obtain a secondary SNS-595 hydrate; and (c)dehydrating the secondary SNS-595 hydrate obtained in step (b) to obtainthe substantially pure SNS-595 substance.
 2. The process of claim 1,wherein the first and second aqueous bases are ethanol; theneutralizations of steps (a) and (b) are accomplished with acetic acid;and the dehydration of steps (b) and (c) are accomplished with ethanol.3. The process of claim 2, wherein the substantially pure SNS-595substance obtained from step (c) comprises about 0 to 0.01% Compound 4

based on total weight of the substantially pure SNS-595 substance. 4.The process of claim 1, wherein Compound 3 is obtained by reaction ofCompound 1

with Compound 2


5. A process for preparing substantially pure Compound 3

comprising reacting Compound 1

with Compound 2

in the presence of N,N-diisopropylethylamine and water to obtainsubstantially pure Compound
 3. 6. The process of claim 5, wherein thesubstantially pure Compound 3 obtained comprises from 0 to about 0.1%Compound 1

based on total weight of substantially pure Compound
 3. 7. A process forpreparing a substantially pure SNS-595 substance, comprising: (a)reacting Compound 1

with Compound 2

in the presence of N,N-diisopropylethylamine and water to obtainsubstantially pure Compound 3

(b) reacting the substantially pure Compound 3 with an aqueous basefollowed by neutralizing to obtain a primary SNS-595 hydrate; and (c)dehydrating the primary SNS-595 hydrate obtained in step (a) to obtainthe substantially pure SNS-595 substance.
 8. The process of claim 7,wherein the substantially pure Compound 3 obtained comprises about 0 to0.1% Compound 1

based on total weight of the substantially pure Compound
 3. 9. Theprocess of claim 7, wherein the substantially pure SNS-595 substanceobtained from step (c) comprises about 0 to 0 01% Compound 4

based on total weight of the substantially pure SNS-595 substance. 10.The process of claim 7, wherein the substantially pure SNS-595 substanceobtained from step (b) comprises about 0 to 0.02% Compound 5

based on total weight of the substantially pure SNS-595 substance.
 11. Acomposition comprising substantially pure SNS-595 substance, wherein thesubstantially pure SNS-595 substance comprises about 0 to 0.03% Compound4

based on total weight of the substantially pure SNS-595 substance. 12.The composition of claim 11, wherein the substantially pure SNS-595substance comprises about 0 to 0.01% Compound 4, at the time ofproduction of the composition.
 13. A composition comprising SNS-595 andabout 0 to 0.04 wt % Compound 5


14. The composition of claim 13, wherein the substantially pure SNS-595substance comprises about 0 to 0.02% Compound 5, at the time ofproduction of the composition.
 15. A composition comprising SNS-595 andwater, wherein about 100 mg of SNS-595 is present for every 10 mL of thecomposition, the composition is essentially free of visible particles,and the composition is stable with respect to visible particles.
 16. Thecomposition of claim 15, wherein the composition consists essentially of100 mg SNS-595, 450 mg D-sorbitol, water, and methanesulfonic acid,wherein the pH of the composition is about 2.5, and wherein the water ispresent in an amount to provide a total composition volume of 10 mL. 17.The composition of claim 11, wherein the composition is stable withrespect to visible particles when contacted with a compound capable oftransforming Compound 4

to Compound 6


18. The composition of claim 15, wherein the composition is stable for1, 3, 6, 9, 12, 18, 24, 36, or 42 months.
 19. A composition comprisingSNS-595 and water, wherein about 100 mg of SNS-595 is present for every10 mL of the composition, and the composition is stable with respect toquantity of sub-visible particles therein.
 20. A composition consistingessentially of SNS-595 and 0.03 wt % or less Compound 4


21. A composition consisting essentially of SNS-595 and 0.04 wt % orless Compound 5


22. A composition comprising SNS-595 and 0.07 wt % or less totalCompound 4 and Compound 5


23. A composition comprising SNS-595 and 0.3 mg or less Compound 4

per gram of SNS-595.
 24. A composition comprising SNS-595 and 0.4 mg orless Compound 5

per gram of SNS-595.
 25. A composition comprising SNS-595 and 0.7 mg orless total Compound 4 and Compound 5

per gram of SNS-595.
 26. A composition comprising SNS-595 and less thanor equal to 0.15% of compound 7

based on total weight of the composition.
 27. An aqueous solutionconsisting essentially of: (a) 10 g of a substantially pure SNS-595substance comprising SNS-595 and Compound 4 wherein the substantiallypure SNS-595 substance contains 3 mg or less Compound 4 per gram

(b) 4.5 g of sorbitol: and (c) sufficient methanesulfonic acid toprovide a pH of 2.5; per 100 mL of the solution.
 28. A productcomprising a container containing 10 mL of the solution of claim
 25. 29.An aqueous solution consisting essentially of: (a) 10 g of asubstantially pure SNS-595 substance consisting of SNS-595 and Compound5

wherein the substantially pure SNS-595 substance contains 4 mg or lessCompound 5 per gram; (b) 4.5 g of sorbitol: and (c) sufficientmethanesulfonic acid to provide a pH of 2.5; per 100 mL of the solution.30. A product comprising a container containing 10 mL of the solution ofclaim
 27. 31. An aqueous solution consisting essentially of: (a) 10 g ofa substantially pure SNS-595 substance consisting of SNS-595, Compound 4and Compound 5

wherein the substantially pure SNS-595 substance contains 7 mg or lesstotal Compound 4 and Compound 5 combined per gram; (b) 4.5 g ofsorbitol: and (c) sufficient methanesulfonic acid to provide a pH of2.5; per 100 mL of the solution.
 32. A product comprising a containercontaining 10 mL of the solution of claim 29.